Hemophilia A is a congenital bleeding disorder caused by genetic mutations affecting a plasma protein, termed factor VIII (fVIII), whose function is to facilitate blood clotting. State of the art treatment of hemophilia A consists of frequent intravenous infusion of fVIII containing products. The current limitations to treating hemophilia are 1) morbidity due to joint disease resulting from repeated bleeding into individual joints, 2) the cost of fVIII products, 3) the development of immune responses against fVIII that hinder treatment efficacy and 4) access to fVIII products. Owing to these limitations approximately 30% of hemophilia A patients are treated worldwide. Gene therapy has the potential to cure hemophilia A since only a limited amount of fVIII is needed to provide clinical benefit to the patient. However, three phase 1 clinical trials have been conducted and the outcome has been disappointing due to the extremely low, non-therapeutic levels of fVIII produced by each gene therapy strategy. The mission of Expression Therapeutics is to develop products that will improve the standard treatment of individuals with hemophilia A by incorporating our technology, which is based on proprietary methods that result in dramatic increases in fVIII biosynthesis and secretion from genetically- engineered cells. Our Phase I STTR preclinical studies demonstrated proof-of-concept that Expression Therapeutics'high expression fVIII technology facilitates very high-level fVIII production in a mouse model of hemophilia A following ex vivo retroviral modification and transplantation of hematopoietic stem cells. These studies lead us to the identification of a lead fVIII transgene, designated ET-801, that is the focus of this proposal. ET-801 contains >90% human sequence and <10% of sequences that confer the high-expression properties required to overcome the hurdle of low-level expression seen in previous clinical trials. The goal of the proposed studies in the current Phase II STTR application is to complete the preclinical manufacturing, efficacy and toxicology studies necessary for IND approval and initiation of a human clinical trial. We propose to manufacture and characterize cGMP grade ET-801 lentiviral vector in vitro and in vivo in order to evaluate the effectiveness of our proposed gene therapy strategy in small (mouse) and large (canine) animal models. Completion of these studies will constitute the framework for which a phase 1 clinical trial will be developed and conducted. PUBLIC HEALTH RELEVANCE: Current treatment for hemophilia A, which is a bleeding disorder caused by genetic mutations affecting a blood protein, termed factor VIII (fVIII), relies on infusion of plasma-derived or recombinant fVIII to restore circulating fVIII activity. This therapy is extremely expensive and difficult to maintain, and is, therefore, only offered to 30% of hemophilia A patients worldwide. Gene therapy can cure the disease and this proposal focuses on overcoming the obstacles that limit successful gene therapy for hemophilia A.